5-Aroylindoles Act as Selective Histone Deacetylase 6 Inhibitors

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5-Aroylindoles Act as Selective Histone Deacetylase 6 Inhibitors Ameliorating Alzheimer’s Disease Phenotypes Hsueh-Yun Lee, Sheng Jun Fan, Fang-I Huang, Hsin-Yi Chao, Kai-Cheng Hsu, Tony Eight Lin, TengKuang Yeh, Mei-Jung Lai, Yu-Hsuan Li, Hsiang-Ling Huang, Chia-Ron Yang, and Jing-Ping Liou J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00151 • Publication Date (Web): 20 Jul 2018 Downloaded from http://pubs.acs.org on July 21, 2018

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Journal of Medicinal Chemistry

Revised manuscript 5-Aroylindoles Act as Selective Histone Deacetylase 6 Inhibitors Ameliorating Alzheimer’s Disease Phenotypes Hsueh-Yun Lee,†,＆ Sheng-Jun Fan,§,＆ Fang-I Huang,§,＆ Hsin-Yi Chao,† Kai-Cheng Hsu,¶ Tony Eight Lin,¶ Teng-Kuang Yeh,# Mei-Jung Lai,‡ Yu-Hsuan Li,† Hsiang-Ling Huang,† Chia-Ron Yang,§,* JingPing Liou†,*

School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei 11031, Taiwan. School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan. Research Center of Cancer Translational Medicine, Taipei Medical University, Taipei, Taiwan. Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan. Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan.

†

School of Pharmacy, College of Pharmacy, Taipei Medical University, 250 Wuxing Street, Taipei 11031, Taiwan.

§

School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan.

‡

Research Center of Cancer Translational Medicine, Taipei Medical University, Taipei, Taiwan.

#

Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhunan Town, Miaoli

County, Taiwan. ¶

Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical

University, Taipei, Taiwan.

ACS Paragon Plus Environment

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ABSTRACT: This paper reports the development of a series of 5-aroylindolyl-substitued hydroxamic acids. N-hydroxy-4-((5-(4-methoxybenzoyl)-1H-indol-1-yl)methyl)benzamide (6) has potent inhibitory selectivity against histone deacetylase 6 (HDAC6) with an IC50 value of 3.92 nM. It decreases not only the level of phosphorylation of tau proteins but also the aggregation of tau proteins. Compound 6 also shows neuroprotective activity by triggering ubiquitination. In animal models, compound 6 is able to ameliorate the impaired learning and memory, and it crosses the blood-brain-barrier after oral administration. Compound 6 can be developed as a potential treatment for Alzheimer’s disease in the future.


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INTRODUCTION In the normal conditions, DNA exists in the “wrapped” form built up by interaction with histone deacetylase (HDAC). During proliferation, the condensed DNA is subject to relaxation that allows the translation process. The “loose” and “condensed” forms of DNA can be determined by the acetylation level of lysine residues. Recently, HDAC has attracted significant scientific attention due to its relationship to epigenetic regulation, which is a process that regulates the genetic expression without affecting the sequence of DNA. As a result, HDAC has been considered to be a feasible target for the treatment of epigenetic aberrance. There are 18 isotypes of HDACs with different specific locations and distinct functions. Recent research has focused on the development of selective HDAC isoform inhibitors and attempts have been made to understand the detailed functions and avoid unexpected side effects. HDAC6 is an example that has attracted much attentions recently due to its connection with a diversity of diseases such as cancer, neurodegeneration, and immune disorders.1 Many HDAC6 inhibitors have been reported.2 For example, ACY1215 (II) was reported to have remarkable selective HDAC6 inhibitory activity and in combination with bortezomib, to inhibit significantly the progress of multiple myeloma. It is undergoing clinical trials (phase I/II) in combination with bortezomib for the treatment of multiple muyeloma.3 Attention has also been drawn to the relationship of HDAC6 to the development of Alzheimer’s disease (AD). The formation of neurofibrillary tangles (NFTs) is highly correlated with hyperphosphorylation of tau protein - a phosphorylation which is modulated by HDAC6.4 It has been found that the increasing


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expression of HDAC6 in the brain of AD patients destroys the posttranscriptional regulation of tubulin, the building block of the microtubule that is the crucial component for axonal transport.5 Consequently, HDAC6 has emerged as an attractive target for neurodegenerative diseases like Alzheimer’s disease. Some examples of HDAC6 inhibitors include Tubastatin A (I), developed by Butler et al. in 2010, which behaves as a selective HDAC6 inhibitor with neuroprotective activity,6 and ACY738 (III) which is also a selective HDAC6 inhibitor which has been shown to recover the Alzheimer’s disease phenotype in an animal model.7 Hydroxamic acid is a significant moiety for HDAC inhibitors. Literature surveys indicate that the “Cap” and “Linker” parts may contribute to the selectivity towards specific HDAC isoforms. As a result, a great deal of research has focused intensively on the modification of these parts which are thought to be related to the selectivity of HDAC isoforms.8 Literature surveys have found that the carbonyl group is commonly utilized in the development of HDAC inhibitors. For example, Mahboobi et al. linked a phenyl ring to indole (1a) and furan (1b) through a carbonyl group.9 Ragno et al. synthesized a series of aroyl-pyrrole-hydroxy-amides (APHAs, 2) and the resulting products showed remarkable selectivity toward HDAC1.10 Similar to the aroylheterocycles mentioned above, we have synthesized a series of 5aroylindoles (3) and found that they show potent antitubulin and antiproliferative activity.11 In an attempt to broaden the known applications of the 5-aroylindole motif, we report in this paper the synthesis of a series of 5-aroylindoly-containing hydroxamic acids (4-20) and an investigation of their biological activities, including HDAC isoform selectivity and their effects on Alzheimer’s disease.


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Figure 1. Structures of HDAC6 inhibitors (I-III).


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Figure 2. The design of 5-aroylindolyl hydroxamic acids (4-20).

RESULTS AND DISCUSSION Chemistry. Scheme 1 illustrates the synthesis of compounds 4-10 without any linkages between hydroxamic

acid

and

benzene

ring.

5-Formylindole

(21)

was

reacted

with

methyl

4-

(chloromethyl)benzoate in the presence of potassium t-butoxide (t-BuOK) to afford compound 22. The reaction of 22 with various Grignard reagents followed by oxidation with pyridinium dichromate (PDC) yielded compound 23. The ester group of 23 was hydrolyzed by LiOH to generate the corresponding carboxylic acid which, subjected to amidation with O-(tetrahydro-pyran-4-yl)-hydroxylamine (NH2OTHP) followed by TFA-medicated deprotection, afforded the designed hydroxamic acids (4-10). Scheme 2 describes the synthetic routes to compounds 11-20. Compound 21 was reacted with 3bromobenzenesulfonyl chloride, 4-bromobenzenesulfonyl chloride, or 4-bromobenzyl bromide in the presence t-BuOK to yield compound 24. The resulting products were reacted with various Grignard reagents followed by oxidation with PDC to afford compounds 25. The resulting products underwent Heck olefination with t-butyl acrylate to afford the corresponding cinnamates 26. The ester group of 26 was hydrolyzed by trifluoroacetic acid (TFA) and this was followed by amidation with O-(tetrahydro2H-pyran-2-yl)hydroxylamine (NH2OTHP) and deprotection by 10% TFA to provide compounds 11-20.

Scheme 1. Synthetic Approaches to Compounds 4-10a ACS Paragon Plus Environment

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a

Reagents and conditions: (a) methyl 4-(chloromethyl)benzoate, t-BuOK, THF, rt; (b) i. Grignard reagents, THF, 0 oC to rt;

ii. PDC, CH2Cl2, molecular sieves, rt; (c) i. LiOH(aq), dioxane, 40 oC; ii. NH2OTHP, EDC·HCl, HOBt, NMM, DMF, rt; iii.

TFA(aq), MeOH, rt.

Scheme 2. Synthetic Approaches to Compounds 11-20a


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a

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Reagents and conditions: (a) potassium tert-butoxide, bromobenzenesulfonyl chlorides or 4-bromobenzyl bromide, DMF,

rt; (b) i. Grignard reagents, THF, 0 oC to rt; ii. PDC, CH2Cl2, molecular sieves, rt; (c) tert-butyl acrylate, Pd2(dba)3, PPh3,

TEA, NaHCO3, DMF, 110 oC; (d) i. TFA, rt; ii. NH2OTHP, EDC·HCl, HOBt, NMM, DMF, rt; iii. TFA(aq), MeOH, rt.

BIOLOGICAL EVALUATION A. Inhibition of HDAC activity and evaluation of HDAC6 selectivity of test compounds. Table 1 illustrates the inhibitory activity of tested compounds (4-20) toward HDAC1, 2, 3, 6, 8, 10 and a reference compound, trichostatin A. The first glance at the result shows that the 4-(Nhydroxyaminocarbonyl)benzyl group of compounds 4-10 is responsible for the increase of HDAC6 ACS Paragon Plus Environment

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inhibitory activity. In addition, compounds 4-10 have distinct selectivity ratio toward HDAC6 over other HDAC isoforms, as compared with compounds 11-20. In the comparison of 6 with 19, the addition of a –C=C– linkage (19) led to a marked decrease of HDAC6 inhibitory activity and a weak HDAC6 selectivity. The replacement of –CH2– group of compound 19 with –SO2– group (17) resulted in a decrease of HDAC isoform inhibitory activity and a slight loss of HDAC6 selectivity. The shift of N-hydroxyacrylamde group (–C=C–CONHOH) from para-position (17) to meta-position (12) resulted in increase of inhibitory activity against HDAC1, 2, 3, 8, and 10; as a result, losing HDAC isoform selectivity. Among all the synthesized compounds, 6 shows the most potent HDAC6 inhibitory activity when compared to other isotypes of HDACs. It inhibits HDAC6 with an IC50 value of 3.92 nM, which is 558.7-, 144.9-, 1058.7-, 164.8-, and 15255.1-fold more selective than HDAC1, 2, 3, 8, and 10, respectively. Further, compound 6, at concentrations ranging from 0.1 µM to 1 M, significantly increases the acetylation of α-tubulin in SH-SY5Y cells and has no effect on the acetylation of histones, which is consistent with its selective inhibition of HDAC6 (Figure 3). In addition, treatment of ACY1215 results in similar levels of acetylation of α-tubulin to that of controls.3

Table 1. Inhibition of the activities (IC50, nMa) of HDAC isoforms 1, 2, 3, 6, 8 and 10; in addition, their selectivity ratio toward HDAC6 (shown in the bracket). Class I HDAC1

HDAC2

Class II HDAC3

HDAC8

Compd

HDAC10 HDAC6

(HDAC1/HDAC6)

(HDAC2/HDAC6)

(HDAC3/HDAC6)

(HDAC8/HDAC6)


9

(HDAC10/HDAC6)


4

3300

5110

4400

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1240

>100000 9.25

5

(356.8)

(552.4)

(475.7)

(134.1)

4780

2100

6620

1060

(>10810.8) >100000 6.73

6

(710.3)

(312.0)

(983.7)

(157.5)

2190

568

4150

646

(>14858.8) 59800 3.92

7

(558.7)

(144.9)

(1058.7)

(164.8)

3790

5140

5090

1120

(15255.1) 47900 16.4

8

(231.1)

(313.4)

(310.4)

(68.3)

2800

3030

6050

756

(2920.7) 18900 15.5

(180.6)

(195.5)

(390.3)

(48.8)

9

NDb

ND

ND

ND

10

3120

7250

3910

1490

(1219.4) 1540

ND >100000

11.9

11

(262.2)

(609.2)

(328.6)

(125.2)

230

1800

59.6

2750

(>8403.4) 5240 1420

12

(0.16)

(1.3)

(0.04)

(1.9)

1460

424

232

812

(3.7) 5890 751

13

(1.7)

(0.6)

(0.3)

(1.1)

1210

4800

323

3530

(7.8) 7740 1810

14

(0.7)

(2.7)

(0.2)

(1.9)

877

2140

861

1420

(4.3) 8240 1180

15

(0.7)

(1.8)

(0.7)

(1.2)

1320

1770

>100000

1260

(6.9) >100000 562

16

(2.3)

(3.1)

(>177.9)

(2.2)

1420

7270

891

4480

(>177.9) 9100 2750

17

(0.5)

(2.6)

(0.3)

(1.6)

7160

>100000

2560

10000

(3.3) >100000 848

18

(8.4)

(>117.9)

(3.01)

(11.8)

8540

11600

996

5340

(>117.9) >100000 972

(8.8)

(11.9)

(1.02)

(5.5)


10

(>102.9)

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Journal of Medicinal Chemistry 19

1440

1950

2050

2810

7050 108

20

(13.3)

(18.1)

3480

5740

(18.98)

(26.01) 2170

ND

Trichostatin

(24.3)

(40.1)

12.0

46.6

(65.3)

143

ND

(15.2) 52.5

566

34.5 3.49

A a

(3.4)

(13.4)

(15.04)

(162.2)

(9.9)

These assays were conducted by the Reaction Biology Corporation, Malvern, PA. All compounds were dissolved

in DMSO and tested in 10-dose IC50 mode with 3-fold serial dilution starting at 10 μM; bND: not determined.


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Figure 3. Inhibition of the acetylation of α-tubulin and histone H3 in SH-SY5Y cells treated with compound 6. SH-SY5T cells were incubated for 24 h with or without the compound 4-20 (1 M), SAHA (3 M) (A), or indicated concentrations of compound 6, ACY-1215, or SAHA (B). Whole-cell lysates


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were subjected to western blotting with the indicated antibodies. The results represent the mean ± STD of three independent experiment; *p < 0.05, **p < 0.01, and ***p < 0.001 compared with controls. SAHA is a pan-HDAC inhibitor approved by FDA for treatment of cutaneous T cell lymphoma.

B. Interaction analysis of compound 6 in class I HDACs and class IIa HDAC6. To elucidate the selectivity mechanism of compound 6, we docked the compound into the available crystal structures of HDAC1 (PDB ID: 5ICN), 2 (PDB ID: 5IX0), 3 (PDB ID: 4A69), 6 (PDB ID: 5EDU) and 8 (PDB ID: 1W22), and then compared the structures. Superposition of the structures revealed a specific pocket of HDAC6, which is created from a loop and a helix (Figure 4A). Residue W497 on the loop yields van der Waals interactions with residue H560 on the helix, which causes the loop to extend towards the helix to form the specific pocket (Figure 4B). The docking result showed that the cap region of compound 6 binds to the specific pocket. The anisole moiety of compound 6 forms van der Waals interactions with residues N494, D496 and W497 and yields a hydrogen bond with residue N494 (Figure 4A). In contrast, HDAC1, 2, 3 and 8 does not have a corresponding pocket on the surface. The lack of the specific pocket of these HDAC isozymes lead to weak inhibitory activity of compound 6 compared to HDAC6 (Table 1). The structure analysis suggests that the anisole moiety and its interactions with the specific pocket are important for the selectivity of compound 6.


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A HDAC1 HDAC2 HDAC3 HDAC6 D496

HDAC8

N494

Compound 6

W497 H560

Zn

B D496

W497

N494

Zn

Zn

Zn

H560

HDAC6

HDAC1

Zn

HDAC3

HDAC2

Zn

HDAC8

Figure 4. (A) Docking pose of compound 6 (yellow) in the binding site of HDAC6 (blue). Residues that create the specific pocket in HDAC6 are represented as a stick and labeled as shown. HDAC1 (purple), 2 (orange), 3 (green) and 8 (pink) are superimposed onto HDAC6. (B) Surface model of respective HDAC isozymes and the docking poses of compound 6 in respective binding sites. The surface comparison reveals a specific pocket only present in HDAC6. The specific pocket is exploited by the


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cap region of compound 6. The zinc ion is represented as a grey sphere and hydrogen bond as a green dotted line.

C. Inhibition of tau phosphorylation and aggregation. The

characteristic

features

of

AD

include

aggregation

of

amyloid

β

(Aβ)

plaques

hyperphosphorylated tau tangles.12 Tau is a microtubule-associated protein that stabilizes axonal microtubules;13 however, in AD, tau aggregates into paired helical filaments that are deposited as neurofibrillary tangles (NFTs).13,14 Further, amyloid β peptides can enhance the accumulation of tau aggregates and NFTs, leading to synaptic dysfunction and eventual neuronal death.12,14,15 Thus, tau phosphorylation was measured in SH-SY5Y and Neuro-2a cells that are treated with A1-40 or transfected with pCAX APP 695 and pRK5-EGFP-Tau P301L plasmids, which respectively encodes tyrosine/histidine mutations of APP and human mutant P301L-tau.16,17 As shown in Figure 5A, phosphorylation on Ser396 significantly increases in cells transfected with P301L, and more tau phosphorylation can be observed in cells cotransfected with P301L and hAPP365 plasmids. Compounds 4, 5, and 6 exhibit the most potent HDAC6 inhibitory activity in this series (Table 1); and therefore, we next sought to determine whether they inhibit tau phosphorylation. Compounds 4, 5, and 6 significantly inhibit phosphorylation of tau Ser396 in SH-SY5Y or Neuro-2a cells, and compound 6 is the most potent inhibitor (Figure 5B). Taken together with other assays such as mouse microsome stability, water solubility, and LogP prediction (Supplemental information Table S1-S3), compound 6


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was used in further studies. In transfected cells, compound 6 markedly inhibits tau phosphorylation on Ser396 and Ser404, slightly reduces APP levels in transfected cells (Figure 5C), and diminishes A1-40induced tau phosphorylation (Figure 5D). Hyperphosphorylation raises the ability of tau molecules to assemble and take shape aggregation to fibrils, eventually leading to form NFTs that cause neuronal dysfunction.18,19 We used a published method20 to evaluate whether compound 6 suppresses the polymerization of phosphorylated tau (Figure 6A). The levels of tau in the cytosol, membranes, and in aggregates significantly rises in hAPP695/d hTauP301L-transfected SH-SY5Y cells (Figure 6B). Treatment with compound 6 results in reduced levels of aggregated and cytolic tau in hAPP695/d hTauP301L-transfected SH-SY5Y cells. Flow cytometry revealed that compound 6 significantly decreases the sub-G1 population of SH-SY5Y cells (Figure 7), suggesting that compound 6 inhibits phosphorylation and aggregation of tau and downregulates tau aggregation involved in apoptosis.


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Figure 5. Compound 6 inhibits tau phosphorylation. (A) SH-SY5Y or Neuro-2a cells were transfected for 24 h with pCAX APP 695 or pRK5-EGFP-Tau P301L, and were then harvested, and cell lysates were subjected to western blotting. (B, C) Cells were transfected for 24 h with pCAX APP695 and pRK-EGFP-Tau P301L plasmids. The cells were then incubated with or without compound 4, 5, or 6 (1 M) (B) or compound 6 (1 M) (C) for a further 24 h. Cell lysates were prepared for western blot analysis using the indicated antibodies. (D) SH-SY5Y cells were incubated with or without A1-40 (10 M) for 24 h and then with compound 6 (1 M) for a further 24 h. Cell lysates were subjected to

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western blot analysis of the indicated proteins. Results are shown as the mean  STD from three independent experiments. ***p < 0.001 compared with control group; ## p < 0.01, ### p < 0.001 compared with indicated groups.

Figure 6. Effect of compound 6 on tau aggregation. SY-SY5Y cells were transfected with pCAX APP 695/pRK5-EGFP-Tau P301L plasmids for 24 h and then incubated with compound 6 (1 M) for a further 24 h. (A) Flow chart of fractionation to separate different parts of tau. (B) Western blot analysis of the effects of compound 6 on tau pools generated using as (A). Results are shown as the mean  STD. *p < 0.05 and **p < 0.01 compared with the corresponding controls.


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Figure 7. Compound 6 significantly inhibits apoptosis of SH-SY5Y cells induced by phosphorylated tau. Flow cytometric analysis of DNA content and the distribution of cells in different stages of the cell cycle. Cells were transfected with pCAX APP 695/pRK5-EGFP-Tau P301L for 24 h, incubated for a further 24 h with or without compound 6 at the indicated concentrations, fixed, and stained using propidium iodide.

D. Neuroprotective effects of compound 6. Hyperphosphorylated tau formed NFTs is a primary pathological component of AD. Expediting the clearance of hyperphosphorylated tau may serve as an effective therapeutic strategy. The significance of tau-chaperone interactions in the pathology of AD is unclear, but increasing evidence suggests that clearance of tau aggregates, which is mediated by the heat shock protein (HSP)–ubiquitin-proteasome ACS Paragon Plus Environment

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system (UPS), plays a pivotal role and underlines Hsp90 as a key mediator of tau degradation.21 Hsp90 is a major heat shock protein that interacts with diverse proteins, including tau.22 After binding Hsp90, a protein enters a refolding pathway or is degraded by the UPS.23,24 Post-translational modifications such as acetylation can affect the function of Hsp90.24,25 For instance, hyperacetylated Hsp90 decreases the affinity of binding of Hsp90 to misfolded protein (e.g. phosphorylated tau) complex, then phosphorylated

tau

was

transferred

to

the

Hsp70/CHIP

complex

and

undergo

further

degradation.23,25,26,27 Hsp90 is an HDAC6 substrate, suggesting that down-regulation of the expression or activity of HDAC6 may promote Hsp90 acetylation, reduce the affinity of binding of Hsp90 to protein complex, increase Hsp70 expression and thereby resulting in tau degradation.23,26,28 Consequently, we explored the neuroprotective mechanism of compound 6 through Hsp–UPS modulation. HDAC6/Hsp90 binding is significantly increased in cotransfected cells (Figure 8A); and acetylation of Hsp90 is increased, which was accompanied by decreased HDAC6/Hsp90 binding and increased Hsp70 expression in response to compound 6 treatment (Figure 8A, 8B). In cells treated with compound 6, co-immunoprecipitation of ubiquitin with phosphorylated tau (Ser396) distinctly increases (Figure 8C), and the level of polyubiquitinated proteins significantly accumulates as well (Figure 8D), suggesting the subsequent proteasomal degradation of polyubiquitinated proteins.


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Figure 8. Compound 6 increases the ubiquitination of phosphorylated-tau. (A, B, C) SH-SY5Y and Neuro-2a cells were transfected with pCAX APP 695/pRK5-EGFP-Tau P301L plasmids for 24 h and incubated with compound 6 or ACY-1215 (1 M) for a further 24 h, then cell lysates were immunoprecipitated with antibodies against HDAC6, acetyl-lysine (A), or ubiquitin (B) and were subjected to immunoblotting; or cell lysates were prepared for western blot analysis of the indicated

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proteins (B). The results are shown as the mean  STD of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with controls. (D) SH-SY5Y cells were transfected for 24 h with pCAX APP 695/pRK5-EGFP-Tau P301L plasmids. Immunoblots show polyubiquitin complexes.

E. Effects of compound 6 on learning and memory. We investigated whether compound 6 ameliorates impaired learning and memory. We used the Morris water maze test to evaluate learning by training rats to use a hidden platform. The scopolaminetreated rats display a prolonged latency to find the platform, which is shortened when they are treated with compound 6 (Figure 9A), and the rats remain longer on the target quadrant (Figure 9B). Moreover, we used the elevated plus maze to assess spatial memory. Rats treated with compound 6 spend less time finding the closed-arm compared with controls (Figure 9C). Immunohistochemical analysis revealed that the scopolamine-mediated increase in tau phosphorylation at Ser396/Ser404 is significantly downregulated in the hippocampal CA1 region (Figure 9D) of rats treated with compound 6. Triple transgenic (3xTg-AD) mice containing APPSwe and tauP301L mutant transgenes were developed neuropathologies such as plaque and tangles,29 followed by further investigation of the neuroprotective effect of compound 6. Compared with controls, oral administration of compound 6 significantly ameliorates memory impairment (Figure 9E). Immunohistochemical analysis the CA1 region in 3xTg-AD mice showed that compound 6 treatment could down regulate the expression of p-Tau (S396), p-Tau (S404), and -amyloid and increase acetyl--tubulin in the mice brain (Figure 9F). Western blot results also ACS Paragon Plus Environment

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indicated compound 6 treatment remarkably inhibited p-Tau (S396), p-Tau (S404) and -amyloid expression and raised acetyl--tubulin levels in 3xTg-AD mice brain (Figure 9G). These results reveal that compound 6 treatments is able to penetrate the BBB and to ameliorate Alzheimer’s deficits.

Figure 9. Compound 6 significantly ameliorates scopolamine-induced spatial memory impairment. Wistar rats (aged 7 weeks) were orally administered compound 6 (25 mg/kg) for 2 weeks, scopolamine (1 mg/kg) was injected (i.p.) 90 min before the test trial, and the rats were then subjected to the Morris

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water maze (A, B) and elevated plus maze (C) models. (A) Representative behavioral traces. (B) Time spent in the target quadrant during the probe test. (C) Time taken by each animal to move from the open arm to a closed arm within 180 s. (D) Immunohistochemical detection of acetyl-α-tubulin levels and tau phosphorylation on Ser396 and Ser404 in the hippocampal CA1 region. Black arrows indicate phosphorylated-tau, and the enlarged images represent the areas indicated by the red arrows. Scale bar = 50 m. (E, F, G) 3xTg-AD mice (six months of age) were orally administered compound 6 (50 mg/kg), memantine (30 mg/kg) (E, F) or compound 6 (50, 100 mg/kg) (G) daily for three months, and the rats were then subjected to the Morris water maze, and measure the escape latency times (E); or the mice were sacrificed and removed the brain to detect the immunohistiochemical analysis for acetyl-α-tubulin levels and tau phosphorylation on Ser396 and Ser404 in the hippocampal CA1 region by immunohistiochemical stains (F) and western blot analysis (G). Red arrowheads show -amyloid and phosphorylated-tau proteins. Scale bar = 25 m. Data represent the mean ± STD. ***p < 0.001 compared with the basal or control group; #p < 0.05, ##p < 0.01, ###p < 0.001 compared to the vehicletreated group.

Transport across the blood-brain barrier (BBB) of a drug is an important requirement in treatment of disorders of the central nervous system. To determine whether compound 6 enters the brain, a single oral dose of compound 6 was administered, and samples of plasma and brain tissues were analyzed using LC-MS/MS. Brain and plasma concentrations of compound 6 were detected after oral


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administration, and the brain/plasma ratio was 1.19 (Table 2) for 1 h and sustained at least 3 h. These results clearly indicate that compound 6 penetrates the BBB and improves learning and memory deficits. Figure 10 summarizes the neuroprotective mechanism of compound 6.

Table 2. Brain and plasma concentrations of compound 6 after oral administration. Dose

Time

Brain Concentration

Plasma Concentration

Brain/Plasma

(mg/kg)

(hr)

(ng/g)

(ng/g)

Ratio

PO

50

1

10.4 ± 1.1

8.5 ± 0.2

1.19

PO

50

3

6.3 ± 0.5

7.3 ± 0.6

0.86

Route


25


Figure 10. Summary of the mechanism of the neuroprotective effect of compound 6.

F. Maximum tolerated dose (MTD) assay of compound 6. The results found that compound 6-treated animals showed the normal increase in body weight as well as control group (Figure 11). However, compound 6 at doses of 100, 500, and 1000 mg/kg/day by intraperitoneal injection for six days was well tolerated in mice.

Control (survival rate:100%) Compound 6 (100 mg/kg, ip, qdx6) (survival rate:100%) Compound 6 (500 mg/kg, ip, qdx6) (survival rate:100%) Compound 6 (1000 mg/kg, ip, qdx6) (survival rate:100%)

30

Body weight (g)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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25

20 10 5 0 1

2

3

4

5

6

7

8

9 10 11 12 13 14 15

Days

Figure 11. Maximum tolerated dose (MTD) of 6. The treated animals were administered compound 6 at doses of 100, 500, and 1000 mg/kg/day by intraperitoneal injection for 6 days and then recovered for 1 week. A group of six mice was injected with vehicle as control. Animals were monitored daily for body weight and survival.


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Conclusion In this study, a series of 5-aroylindolyl hydroxamic acids (4-20) were synthesized. Biological assays revealed that compounds 4, 5, and 6 possess potent activity against HDAC6 with IC50 values in the range of single-digit nM. In view of the relationship between HDAC6 and Alzheimer’s disease, compounds 4, 5, and 6 were tested for their effects on Alzheimer’s disease phenotypes. The results indicate that compound 6 is able to reduce tau phosphorylation and aggregation, which is high correlation with the formation of neurofibrillary tangles. Its HDAC6 inhibitory activity led to the increase of acetylated Hsp90, which in turn decreased the binding of Hsp90 and HDAC6, and phosphorylate tau was transferred to the Hsp70/CHIP complex then leading to the ubiquitination of phosphorylate tau proteins. In in vivo experiments, compound 6 helped mice spend less time finding the platform and the closed-arm. In addition, 6 decreased the levels of phosphorylated tau in the CA1 region of hippocampus, which relates to learning and memory.30 Compound 6 is able to cross blood-brainbarrier after oral administration, which is a crucial issue for use of HDAC6 inhibitors as an Alzheimer’s disease treatment. In summary, the ability of compound 6 to improve Alzheimer’s disease phenotypes including tau hyperphosphorylation and aggregation, neuroprotective effects upon ubiquitination, and amelioration of learning and memory deficits, makes it a potential agent for further development as a treatment for Alzheimer’s disease.

EXPERIMENTAL SECTION


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(A) Chemistry. Nuclear magnetic resonance (1H NMR and 13C NMR) spectra were recorded with Bruker Fourier 300 NMR spectrometers, with chemical shifts in parts per million (δ) downfield from TMS, the internal standard. High-resolution mass spectra (HRMS) were recorded with a JEOL (JMS-700) electron impact (EI) mass spectrometer. The purities of the final compounds were determined using an Agilent 1100 series HPLC system using a C-18 column (Agilent ZORBAX Eclipse XDB-C18 5 μm, 4.6 mm × 150 mm). Elution conditions: Mobile phase A-Acetonitrile; Mobile phase B-Water containing 0.1% formic acid + 10 mmol NH4OAc. The flow-rate was 0.2 mL/min, the injection volume was 5 μL, the system operated at 25 °C, and peaks were detected at 210 nm. The purities of the final compounds were found to be ≥ 95%. Flash column chromatography was conducted using silica gel (Merck Kieselgel 60, No. 9385, 230−400 mesh ASTM). All reactions were conducted under an atmosphere of dry N2. 4-[5-(4-Fluoro-benzoyl)-indol-1-ylmethyl]-N-hydroxy-benzamide (4) The title compound was obtained in 35% overall yield from compound 22 in a manner similar to that described for the preparation of 6: mp = 169.6-171.2 oC; 1H NMR (300 MHz, DMSO) δ 5.55 (s, 2H), 6.70 (d, J = 3.0 Hz, 1H), 7.28 (d, J = 8.1 Hz, 2H), 7.36 (t, J = 8.7 Hz, 2H), 7.60 (s, 2H), 7.67-7.73 (m, 3H), 7.77-7.83 (m, 2H), 8.01 (s, 1H), 9.04 (s, 1H), 11.18 (s, 1H). 13C NMR (75 MHz, DMSO) δ 48.99, 103.32, 110.30, 115.23, 115.53, 123.00, 124.59, 126.98, 127.27, 127.67, 128.54, 131.22, 132.09, 132.15, 132.27, 134.99, 135.03, 138.07, 140.94, 162.53, 163.94, 165.83, 194.57. HRMS (ESI) for C23H18FN2O3 (M+H+) calcd 389.1301, found 389.1292. HPLC purity of 99.84% (retention time = 28.45).


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4-[5-(4-Chloro-benzoyl)-indol-1-ylmethyl]-N-hydroxy-benzamide (5) The title compound was obtained in 30% overall yield from compound 22 in a manner similar to that described for the preparation of 6: mp = 156.6-158.7 oC; 1H NMR (300 MHz, DMSO) δ 5.55 (s, 2H), 6.70 (d, J = 3.0 Hz, 1H), 7.26 (d, J = 8.1 Hz, 2H), 7.57-7.64 (m, 4H), 7.67-7.75 (m, 5H), 8.01 (s, 1H), 9.01 (s, 1H), 11.14 (s, 1H). 13C NMR (75 MHz, DMSO) δ 48.95, 80.57, 103.36, 110.35, 122.93, 124.72, 126.94, 127.23, 127.65, 128.24, 128.47, 131.22, 132.05, 136.60, 137.20, 138.12, 140.91, 163.89, 194.74. HRMS (ESI) for C23H16N2O3Cl (M-H+) calcd 403.0849, found 403.0853. HPLC purity of 99.60% (retention time = 31.47). N-Hydroxy-4-[5-(4-methoxy-benzoyl)-indol-1-ylmethyl]-benzamide (6) A mixture of compound 23c (1.0 g, 2.5 mmol) dissolved in dioxane (20 mL), and 1N LiOH (5 mL) was heated at 40 oC for 3 h. To the reaction was added H2O (50 mL) followed by acidification with 3N HCl and extraction with EtOAc (50 mL x 3). The organic layers were collected and purified by column chromatography to afford a white solid. The resulting product was dissolved in DMF (3 mL), and EDC·HCl (0.55 g, 2.87 mmol), HOBt (0.45 g, 2.94 mmol), NMM (0.3 mL, 2.72 mmol) and NH2OTP (0.33 g, 2.81 mmol) were added. After stirring at room temperature for 1 h, the reaction was quenched with H2O and extracted with EtOAc (50 mL x 3). The combined organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure. To the residue was added 10% TFA in MeOH (20 mL) and the mixture was stirred at room temperature for 3 h. The reaction mixture was quenched with H2O and extracted with EtOAc (30 mL x 3). The organic layer was collected and purified by column

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chromatography to afford 6 (0.66 g, 66%). mp = 148.7-149.3 oC; 1H NMR (300 MHz, DMSO) δ 3.85 (s, 3H), 5.54 (s, 2H), 6.68 (d, J = 3.3 Hz, 1H), 7.07 (d, J = 8.7 Hz, 2H), 7.28 (d, J = 8.4 Hz, 2H), 7.50-7.62 (m, 2H), 7.65-7.77 (m, 5H), 7.99 (s, 1H), 9.03 (s, 1H), 11.17 (s, 1H).

13

C NMR (75 MHz, DMSO) δ

48.96, 55.48, 103.11, 110.11, 113.66, 122.96, 124.06, 126.98, 127.26, 127.58, 129.25, 130.73, 131.03, 131.92, 132.07, 137.78, 141.00, 162.31, 163.93, 194.68. HRMS (ESI) for C24H19N2O4 (M-H+) calcd 399.1345, found 399.1345. HPLC purity of 95.27% (retention time = 26.65). N-Hydroxy-4-[5-(3-methoxy-benzoyl)-indol-1-ylmethyl]-benzamide (7) The title compound was obtained in 50% overall yield from compound 22 in a manner similar to that described for the preparation of 6: 1H NMR (300 MHz, DMSO) δ 3.79 (s, 3H), 5.45 (s, 2H), 7.12-7.25 (m, 5H), 7.34-7.41 (m, 3H), 7.59-7.67 (m, 3H), 8.04 (d, J = 1.2 Hz, 1H). 13C NMR (75 MHz, DMSO) δ 50.50, 55.83, 104.65, 110.81, 115.60, 118.78, 123.32, 124.74, 126.42, 128.02, 128.52, 129.48, 130.20, 130.27, 131.71, 132.78, 140.01, 141.35, 142.75, 160.87, 167.61, 199.05. HRMS (ESI) for C24H21N2O4 (M+H+) calcd 401.1501, found 401.1496. HPLC purity of 97.77% (retention time = 30.68). 4-[5-(3,4-Dimethoxy-benzoyl)-indol-1-ylmethyl]-N-hydroxy-benzamide (8) The title compound was obtained in 46% overall yield from compound 22 in a manner similar to that described for the preparation of 6: mp = 116.4-117.7 oC; 1H NMR (300 MHz, DMSO) δ 3.80 (s, 3H), 3.85 (s, 3H), 5.54 (s, 2H), 6.69 (d, J = 3.0 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H), 7.28-7.38 (m,4H), 7.587.73 (m,4H), 8.02 (s, 1H), 11.18 (s, 1H).

13

C NMR (75 MHz, DMSO) δ 48.99, 55.54, 55.72, 103.15,

110.08, 110.58, 112.17, 123.04, 124.11, 124.47, 127.03, 127.28, 127.58, 129.30, 129.73, 130.68, 131.00,


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132.08, 137.79, 141.01, 148.55, 152.24, 194.70. HRMS (ESI) for C25H23N2O5 (M+H+) calcd 431.1607, found 431.1608. HPLC purity of 98.79% (retention time = 28.3). N-Hydroxy-4-[5-(3,4,5-trimethoxy-benzoyl)-indol-1-ylmethyl]-benzamide (9)

The title compound was obtained in 34% overall yield from compound 22 in a manner similar to that described for the preparation of 6: mp = 131.9-132.8 oC; 1H NMR (300 MHz, DMSO) δ 3.77-3.80 (m, 9H), 5.58 (s, 2H), 6.72 (s, 1H), 7.01 (s, 2H), 7.31 (d, J = 7.5 Hz, 2H), 7.60-7.66 (m, 3H), 7.89 (d, J = 7.2 Hz, 2H), 8.08 (s, 1H).

13

C NMR (75 MHz, DMSO) δ 48.99, 56.04, 60.17, 103.44, 107.14, 110.15,

123.12, 124.64, 127.09, 127.67, 128.71, 129.71, 130.06, 131.08, 133.68, 138.04, 140.67, 142.80, 152.51, 167.01, 194.87. HRMS (ESI) for C24H21N2O4 (M+H+) calcd 401.1501, found 401.1496. HPLC purity of 97.17% (retention time = 31.25). N-Hydroxy-4-[5-(4-methyl-benzoyl)-indol-1-ylmethyl]-benzamide (10)

The title compound was obtained in 26% overall yield from compound 22 in a manner similar to that described for the preparation of 6: mp = 157.2-159.0 oC; 1H NMR (300 MHz, DMSO) δ 2.38 (s, 3H), 5.55 (s, 2H), 6.69 (d, J = 3.0 Hz, 1H), 7.28 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.1 Hz, 2H), 7.60-7.68 (m, 5H), 7.72 (d, J = 8.1 Hz, 2H), 8.01 (s, 1H), 9.06 (s, 1H), 11.20 (s, 1H). 13C NMR (75 MHz, DMSO) δ 21.12, 48.98, 103.26, 110.19, 123.00, 124.46, 126.97, 127.29, 127.63, 128.89, 128.92, 129.63, 131.10, 132.09, 135.76, 137.95, 140.99, 142.00, 163.96, 195.61. HRMS (ESI) for C24H21N2O3 (M+H+) calcd 385.1552, found 385.1544. HPLC purity of 98.47% (retention time = 29.13). 3-{3-[5-(4-Fluoro-benzoyl)-indole-1-sulfonyl]-phenyl}-N-hydroxy-acrylamide (11) ACS Paragon Plus Environment

31


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The title compound was obtained in 19% overall yield from compound 24a in a manner similar to that described for the preparation of 19: mp = 172.4-173.6 oC; 1H NMR (300 MHz, DMSO) δ 6.58 (d, J = 15.9 Hz, 1H), 7.01 (d, J = 3.6 Hz, 1H), 7.36 (t, J = 8.7 Hz, 2H), 7.58 (d, J = 20.4 Hz, 1H), 7.64 (t, J = 8.1 Hz, 1H), 7.76-7.84 (m, 3H), 7.91 (d, J = 7.8 Hz, 1H), 7.98-8.03 (m, 3H), 8.14 (d, J = 8.7 Hz, 1H), 8.26 (s, 1H).

13

C NMR (75 MHz, DMSO) δ 110.29, 113.26, 115.48, 115.78, 122.29, 124.45, 125.66,

126.16, 127.17, 128.51, 130.19, 130.76, 132.46, 132.50, 132.63, 133.16, 134.00, 134.04, 136.25, 136.82, 137.69, 166.24, 194.13. HRMS (ESI) for C24H16FN2O5S (M+H+) calcd 463.0764, found 463.0768. HPLC purity of 99.58% (retention time = 31.72). N-Hydroxy-3-{3-[5-(4-methoxy-benzoyl)-indole-1-sulfonyl]-phenyl}-acrylamide (12) The title compound was obtained in 28% overall yield from compound 24a in a manner similar to that described for the preparation of 19: mp = 138.6-140.2 oC; 1H NMR (300 MHz, DMSO) δ 3.83 (s, 3H), 6.61 (d, J = 16.2 Hz, 1H), 6.99-7.01 (m, 3H), 7.54 (d, J = 15.9 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.72 (d, J = 7.72 Hz, 2H), 7.89−8.01 (m, 5H), 8.13 (d, J = 8.4 Hz, 1H), 8.27 (s, 1H). 13C NMR (75 MHz, DMSO) δ 55.54, 110.22, 113.09, 113.83, 122.28, 123.93, 125.61, 126.04, 127.12, 128.34, 129.74, 130.08, 130.69, 132.17, 133.12, 133.28, 135.94, 136.06, 136.78, 137.72, 162.06, 162.81, 194.06. HRMS (ESI) for C26H21N2O7S (M-H+) calcd 505.1069, found 505.1072. HPLC purity of 97.67% (retention time = 30.65).

N-Hydroxy-3-{3-[5-(3-methoxy-benzoyl)-indole-1-sulfonyl]-phenyl}-acrylamide (13)


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The title compound was obtained in 31% overall yield from compound 24a in a manner similar to that described for the preparation of 19: mp = 132.0-133.4 oC; 1H NMR (300 MHz, DMSO) δ 3.79 (s, 3H), 6.59 (d, J = 14.4 Hz, 1H), 7.01 (s, 1H), 7.20-7.30 (m, 3H), 7.40-7.70 (m, 3H), 7.80 (d, J = 8.4 Hz, 1H), 7.90-8.01 (m, 4H), 8.15 (d, J = 7.2 Hz, 1H), 8.26 (s, 1H). 13C NMR (75 MHz, DMSO) δ 55.32, 110.27, 113.17, 114.08, 118.30, 122.03, 122.30, 124.48, 125.58, 126.14, 127.08, 128.41, 129.63, 130.12, 130.68, 132.45, 133.09, 135.85, 136.22, 136.78, 137.65, 138.85, 159.13, 195.09. HRMS (ESI) for C26H19N2O6S (M-H+) calcd 4750964, found 475.0966. HPLC purity of 96.77% (retention time = 30.81). 3-{3-[5-(3,4-Dimethoxy-benzoyl)-indole-1-sulfonyl]-phenyl}-N-hydroxy-acrylamide (14) The title compound was obtained in 23% overall yield from compound 24a in a manner similar to that described for the preparation of 19: mp = 138.7−140.2 oC; 1H NMR (300 MHz, DMSO) δ 3.79 (s, 3H), 3.84 (s, 3H), 6.60 (d, J = 15.9 Hz, 1H), 7.00 (d, J = 3.3 Hz, 1H), 7.05 (d, J = 8.4 Hz, 1H), 7.29 (d, J = 8.4 Hz, 1H), 7.35 (s, 1H), 7.53 (d, J = 15.9 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.90 (d, J = 7.5 Hz, 1H), 7.97−8.02 (m, 3H), 8.13 (d, J = 8.7 Hz, 1H), 8.27 (s, 1H). 13C NMR (75 MHz, DMSO) δ 55.51, 55.76, 110.23, 110.64, 111.98, 113.03, 122.26, 123.93, 124.90, 125.59, 126.07, 127.11, 128.31, 129.65, 130.07, 130.68, 133.11, 133.31, 135.91, 136.78, 137.69, 148.63, 152.75, 194.07. HRMS (ESI) for C26H23N2O7S (M+H+) calcd 507.1226, found 507.1227. HPLC purity of 98.79% (retention time = 28.35). N-Hydroxy-3-{3-[5-(3,4,5-trimethoxy-benzoyl)-indole-1-sulfonyl]-phenyl}-acrylamide (15)

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The title compound was obtained in 25% overall yield from compound 24a in a manner similar to that described for the preparation of 19: mp = 152.0-152.7 oC; 1H NMR (300 MHz, DMSO) δ 3.76 (s, 3H), 3.77 (s, 6H), 6.60 (d, J = 15.9 Hz, 1H), 7.00-7.03 (m, 3H), 7.54 (d, J = 15.9 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.82 (dd, J = 1.5, 8.7 Hz, 1H), 7.90 (d, J = 7.8 Hz, 1H), 7.96-8.03 (m, 2H), 8.07 (d, J = 1.2 Hz, 1H), 8.15 (d, J = 8.7 Hz, 1H), 8.28 (s, 1H), 9.19 (s, 1H), 10.87 (s, 1H). 13C NMR (75 MHz, DMSO) δ 56.04, 60.19, 107.35, 110.39, 113.17, 122.27, 124.51, 125.69, 126.30, 127.17, 128.35, 130.21, 130.73, 132.56, 132.66, 133.15, 136.05, 136.17, 136.80, 137.69, 141.21, 152.62, 162.06, 194.37. HRMS (ESI) for C27H25N2O8S (M+H+) calcd 537.1332, found 537.1336. HPLC purity of 97.37% (retention time = 29.66). N-Hydroxy-3-{3-[5-(4-methyl-benzoyl)-indole-1-sulfonyl]-phenyl}-acrylamide (16) The title compound was obtained in 18% overall yield from compound 24a in a manner similar to that described for the preparation of 19: mp = 129.7-131.1 oC; 1H NMR (300 MHz, DMSO) δ 2.35 (s, 3H), 6.61 (d, J = 15.9 Hz, 1H), 6.99 (d, J = 3.6 Hz, 1H), 7.30 (d, J = 8.1 Hz, 2H), 7.52-7.66 (m, 4H), 7.77 (dd, J = 1.2, 8.7 Hz, 1H), 7.90 (d, J = 7.8 Hz, 1H), 7.98-8.02 (m, 3H), 8.14 (d, J = 8.7 Hz, 1H), 8.27 (s, 1H), 9.19 (s, 1H), 10.87 (s, 1H).

13

C NMR (75 MHz, DMSO) δ 21.12, 110.26, 113.16, 122.28, 124.27,

125.60, 126.11, 127.11, 128.38, 129.05, 129.81, 130.12, 130.68, 132.86, 133.12, 134.70, 136.05, 136.12, 136.80, 137.72, 142.81, 162.06, 195.07. HRMS (ESI) for C25H19N2O5S (M-H+) calcd 459.1015, found 459.1019. HPLC purity of 98.56% (retention time = 33.11). N-Hydroxy-3-{4-[5-(4-methoxy-benzoyl)-indole-1-sulfonyl]-phenyl}-acrylamide (17)


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The title compound was obtained in 24% overall yield from compound 24b in a manner similar to that described for the preparation of 19: mp = 138.7−140.2 oC; 1H NMR (300 MHz, DMSO) δ 3.84 (s, 3H), 6.56 (d, J = 15.6 Hz, 1H), 6.99 (d, J = 3.6 Hz, 1H), 7.06 (d, J = 8.7 Hz, 2H), 7.46 (d, J = 15.9 Hz, 1H), 7.71-7.79 (m, 5H), 7.95-7.98 (m, 2H), 8.04-8.12 (m, 3H), 9.21 (br, 1H), 10.86 (br, 1H). 13C NMR (75 MHz, DMSO) δ 55.55, 110.22, 113.04, 113.83, 123.45, 123.90, 125.99, 127.47, 128.28, 128.67, 129.72, 130.08, 132.16, 133.24, 135.92, 136.68, 141.05, 162.79, 194.06. HRMS (ESI) for C25H19N2O6S (M-H+) calcd 475.0964, found 475.0966. HPLC purity of 98.75% (retention time = 31.00). N-Hydroxy-3-{4-[5-(3,4,5-trimethoxy-benzoyl)-indole-1-sulfonyl]-phenyl}-acrylamide (18)

The title compound was obtained in 28% overall yield from compound 24b in a manner similar to that described for the preparation of 19: mp = 142.4-142.8 oC; 1H NMR (300 MHz, DMSO) δ 3.76-3.79 (m, 9H), 6.55 (d, J = 15.9 Hz, 1H), 7.00-7.03 (m, 3H), 7.45 (d, J = 15.9 Hz, 1H), 7.76-7.83 (m, 3H), 7.96 (d, J = 3.6 Hz, 1H), 8.05-8.08 (m, 3H), 8.11 (d, J = 8.7 Hz, 1H), 9.15 (s, 1H), 10.88 (s, 1H). 13C NMR (75 MHz, DMSO) δ 56.08, 60.22, 107.39, 110.44, 113.21, 123.49, 124.58, 126.30, 127.56, 128.33, 128.73, 130.23, 132.60, 132.70, 136.23, 136.73, 141.12, 141.26, 152.65, 161.85, 194.41. HRMS (ESI) for C27H23N2O8S (M-H+) calcd 535.1175, found 535.1179. HPLC purity of 96.80% (retention time = 29.96). N-Hydroxy-3-{4-[5-(4-methoxy-benzoyl)-indol-1-ylmethyl]-phenyl}-acrylamide (19) A mixture of compound 26i (0.5 g, 1.1 mmol) and TFA (5.0 mL) was stirred at room temperature for 30 min. The reaction was quenched by H2O and extracted with EtOAc (30 mL x 3). The organic layer was collected, basified by aqueous NaHCO3, and dried in vacuo to afford an oily residue. The resulting

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product was dissolved DMF (5.0 mL), and NH2OTHP (0.14 g, 1.2 mmol), EDC·HCl (0.25 g, 1.3mmol), HOBt (0.15 g, 1.1 mmol), and NMM (0.12 mL, 1.1 mmol) were added to the solution; then the mixture was stirred at room temperature for 3 h. The reaction was quenched with H2O and extracted with EA (30 mL x 3). The organic layer was collected and dried in vacuo to obtain an oily product. To the resulting product was added 10% TFA in MeOH (20 mL), and then the resulting mixture was stirred at room temperature for 3 h. The reaction mixture was quenched with H2O and extracted with EtOAc (30 mL x 3). The organic layer was collected and purified by column chromatography to afford 19 (0.12 g, 26%). mp = 145.7−146.3 oC; 1H NMR (300 MHz, DMSO) δ 3.83 (s, 3H), 5.50 (s, 2H), 6.44 (d, J = 15.6 Hz, 1H), 6.67 (d, J = 3.0 Hz, 1H), 7.05 (d, J = 9.0 Hz, 2H), 7.24 (d, J = 8.1 Hz, 2H), 7.43 (d, J = 15.6 Hz, 1H), 7.51 (d, J = 8.1 Hz, 2H), 7.56-7.7.65 (m, 3H), 7.72 (d, J = 8.7 Hz, 2H), 7.99 (s, 1H), 10.80 (s, 1H).

13

C NMR (75 MHz, DMSO) δ 49.10, 55.52, 103.12, 110.16, 113.71, 119.18, 123.03, 124.11,

127.66, 127.87, 129.28, 130.78, 131.05, 131.98, 134.19, 137.86, 139.24, 162.37, 162.75, 194.79. HRMS (ESI) for C26H23N2O4 (M+H+) calcd 427.1658, found 427.1657. HPLC purity of 98.75% (retention time = 31.0). N-Hydroxy-3-{4-[5-(3,4,5-trimethoxy-benzoyl)-indol-1-ylmethyl]-phenyl}-acrylamide (20)

The title compound was obtained in 39% overall yield from compound 24c in a manner similar to that described for the preparation of 19: mp = 136.7−137.2 oC; 13C NMR (75 MHz, DMSO) δ 49.09, 56.07, 60.23, 103.38, 107.18, 110.25, 119.18, 123.13, 124.71, 127.72, 127.88, 128.70, 131.07, 133.75, 134.20,


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137.96, 138.05, 139.21, 140.70, 152.56, 162.79, 194.97. HRMS (ESI) for C28H27N2O6 (M+H+) calcd 487.1869, found 487.1869. HPLC purity of 98.49% (retention time = 33.14). Methyl 4-((5-formyl-1H-indol-1-yl)methyl)benzoate (22) A mixture of compound 21 (2.0 g, 13.77 mmol), t-BuOK (1.7 g, 15.16 mmol) and THF (110 mL) stirred for 40 min. Then methyl 4-(chloromethyl)benzoate (3.05 g. 16.52 mmol) was added to the mixture and stirred for 12 h. The reaction was quenched with H2O and extracted with CH2Cl2 (100 mL x 3). The organic layer was collected, dried over anhydrous MgSO4, and concentrated in vacuo to yield an oily product. The residue was purified by flash column chromatography over silica gel (EtOAc: n-hexane = 1: 8) to afford 22 (2.14 g, 53 %) as a white solid. 1H NMR (300 MHz, CDCl3) δ 3.89 (s, 3H), 5.41 (s, 2H), 6.74 (d, J =3.6 Hz, 1H), 7.13 (s, 1H), 7.15 (s, 1H), 7.23 (d, J = 3.3 Hz, 1H), 7.31 (d, J = 8.7 Hz, 1H), 7.74 (d, J = 9.9 Hz, 1H), 7.96 (dd, J = 1.8, 1.8 Hz, 1H), 7.98 (dd, J = 1.8, 1.8 Hz, 1H) 8.18 (s, 1H), 10.02 (s, 1H). Methyl 4-((5-(4-methoxybenzoyl)-1H-indol-1-yl)methyl)benzoate (23c) To a solution of compound 22 (1.0 g, 3.41 mmol) in THF (50 mL) was added 4methoxyphenylmagnesium bromide (7.0 mL, 6.82 mmol) at 0 oC, and the resulting solution was allowed to stir at room temperature for 2 h. The reaction mixture was quenched with H2O and extracted with CH2Cl2 (30 mL x 3). The organic layer was collected, dried over anhydrous MgSO4, and concentrated in vacuo to yield an oily product. The resulting product was dissolved in CH2Cl2 (60 mL), then PDC (1.4 g, 3.75 mmol) and molecular sieves (1.4 g) were added and stirred at room temperature for 1 h. The

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reaction mixture was filtered through a Celite-silica gel–Celite packed funnel, and the filtrate was purified by flash column over silica gel (EtOAc: n-hexane = 1:4) to afford 23c (1.16 g, 85%) as a yellow solid. 1H NMR (300 MHz, CDCl3) δ 3.89 (s, 3H), 3.90 (s, 3H), 5.42 (s, 2H), 6.67 (d, J = 3.3 Hz, 1H), 6.94 (s, 1H), 6.98 (s, 1H), 7.14 (s, 1H), 7.17 (s, 1H), 7.21 (d, J = 3.3 Hz, 1H), 7.28 (d, J = 8.7 Hz, 1H), 7.70 (s, 1H), 7.82 (dd, J = 2.1, 3.3 Hz, 1H), 7.85 (dd, J = 3.0, 3.3 Hz, 1H), 7.97 (s, 1H),7.99 (s, 1H), 8.12 (d, J = 1.5 Hz, 1H). 1-((3-Bromophenyl)sulfonyl)-1H-indole-5-carbaldehyde (24a) The title compound was obtained in 82% overall yield from compound 21 with 3bromobenzenesulfonyl chloride in a manner similar to that described for the preparation of 22: 1H NMR (300 MHz, CDCl3) δ 6.83 (d, J = 3.9 Hz, 1H), 7.35 (dd, J = 7.8, 8.1 Hz, 1H), 7.65 (d, J = 6.0 Hz, 1H), 7.67-7.91 (m, 3H), 8.03 (dd, J = 1.8, 2.1 Hz, 1H), 8.09-8.10 (m, 2H), 10.05 (s, 1H). 1-((4-Bromophenyl)sulfonyl)-1H-indole-5-carbaldehyde (24b) The title compound was obtained in 90% overall yield from compound 21 with 4bromobenzenesulfonyl chloride in a manner similar to that described for the preparation of 22: 1H NMR (300 MHz, CDCl3) δ 6.80 (d, J = 4.2 Hz, 1H), 7.58 (dd, J = 1.8, 2.4 Hz, 1H), 7.61 (dd, J = 2.1, 2.1 Hz, 1H), 7.64 (d, J = 3.9 Hz, 1H), 7.74 (dd, J = 2.1, 2.4 Hz, 1H), 7.77 (dd, J = 2.1, 2.1 Hz, 1H), 7.87 (d, J = 9.9 Hz, 1H), 8.07 (d, J = 9.6 Hz, 2H), 10.04 (s, 1H). 1-(4-Bromobenzyl)-1H-indole-5-carbaldehyde (24c)


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The title compound was obtained in 88% overall yield from compound 21 with 4-bromobenzyl bromide in a manner similar to that described for the preparation of 22: 1H NMR (300 MHz, CDCl3) δ 5.3 (s, 2H), 6.72 (d, J = 3.3 Hz, 1H), 6.95 (s, 1H), 7.20 (d, J = 3.3 Hz, 1H), 7.32 (d, J = 8.7 Hz, 1H), 7.41 (s, 1H), 7.44 (s, 1H), 7.74 (d, J = 9.9 Hz, 1H), 8.17 (s, 1H), 10.02 (s, 1H). [1-(3-Bromo-benzenesulfonyl)-1H-indol-5-yl]-(4-methoxy-phenyl)-methanone (25b) The title compound was obtained in 65% overall yield from compound 24a in a manner similar to that described for the preparation of 23c: 1H NMR (300 MHz, CDCl3) δ 3.89 (s, 3H), 6.77 (d, J = 3.6 Hz, 1H), 6.96 (d, J = 9.0 Hz, 2H), 7.34 (t, J = 8.1 Hz, 1H), 7.62 (d, J = 3.9 Hz, 1H), 7.67-7.71 (m, 1H), 7.787.84 (m, 4H), 7.96 (d, J = 1.5 Hz, 1H), 8.03-8.05 (m, 2H). (1-(4-Bromobenzyl)-1H-indol-5-yl)(4-methoxyphenyl)methanone (25i) The title compound was obtained in 72% overall yield from compound 24c in a manner similar to that described for the preparation of 23c: 1H NMR (300 MHz, CDCl3) δ 4 6.70 (d, J = 3.6 Hz, 1H), 7.25 (d, J = 7.5 Hz, 1H), 7.34 (t, J = 7.7 Hz, 1H), 7.53 (d, J = 7.8 Hz, 1H), 7.57 (d, J = 3.7 Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H), 8.00 (d, J = 8.4 Hz, 1H), 8.03 (d, J = 7.6 Hz, 1H), 8.10 (d, J = 7.9 Hz, 1H), 8.36 (s, 1H), 9.99 (s, 1H). 3-{3-[5-(4-Methoxy-benzoyl)-indole-1-sulfonyl]-phenyl}-acrylic acid t-butyl ester (26b) The title compound was obtained in 63% overall yield from compound 25b in a manner similar to that described for the preparation of 26i: 1H NMR (300 MHz, CDCl3) δ 1.52 (s, 9H), 3.87 (s, 3H), 6.38 (d, J

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= 16.2 Hz, 1H), 6.75 (dd, J = 0.9, 3.9 Hz, 1H), 6.95 (d, J = 9.0 Hz, 2H), 7.40-7.54 (m, 2H), 7.64-7.68 (m, 2H), 7.76-7.87 (m, 5H), 7.95-8.05 (m, 2H), 8.05 (d, J = 8.7 Hz, 1H). t-Butyl (E)-3-(4-((5-(4-methoxybenzoyl)-1H-indol-1-yl)methyl)phenyl)acrylate (26i) A mixture of compound 25i (0.5 g, 1.07 mmol), t-butyl acrylate (0.5 mL, 1.28 mmol), triethylamine (0.7 mL, 1.07 mmol), tris(dibenzylideneacetone)dipalladium (0.25 mg, 0.27 mmol), tri-t-butylphosphonium tetrafluoroborate (0.38 g, 0.54 mmol), NaHCO3 (0.16 g, 1.07 mmol) and DMF (3 mL) was heated to 110 oC for 5 h. The mixture was filtered off and water was added, followed by extraction with CH2Cl2 (30 mL x 3). The organic layer was collected and dried over anhydrous MgSO 4 and concentrated in vacuo to yield an oily product. The residue was purified by flash column over silica gel (EtOAc: nhexane = 1: 8) to afford 26i (0.38 g, 69%) as a yellow oil. 1H NMR (300 MHz, CDCl3) δ 1.52 (s, 9H), 3.87 (s, 3H), 5.34 (s, 2H), 6.33 (d, J = 15.9 Hz, 1H), 6.64 (d, J =3.6 Hz, 1H), 6.94 (dd, J = 2.1, 2.7 Hz, 1H), 6.96 (dd, J = 1.8, 3.0 Hz, 1H), 7.07 (s, 1H), 7.10 (s, 1H), 7.19 (d, J = 3.3 Hz, 1H), 7.28 (dd, J = 2.7, 4.8 Hz, 1H), 7.41 (s, 1H), 7.44 (s, 1H), 7.54 (d, J = 16.2 Hz, 1H), 7.70 (d, J = 10.2 Hz, 1H), 7.81 (s, 1H), 7.84 (s, 1H), 8.11 (d, J = 1.5 Hz, 1H). (B) Biology. Cell lines. The human neuroblastoma cell line SH-SY5Y, kindly provided by Fan-Lu Kung (School of Pharmacy, National Taiwan University), was maintained in Ham’s F12 nutrient mixture/Minimum essential media with 10% fetal bovine serum, penicillin (100 units/mL) and streptomycin (100 μg/mL). The mouse neuroblastoma cell line Neuro-2a, purchased from the Bioresource Collection and Research


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Center (Hsinchu, Taiwan) was cultured in Minimum essential media containing 10% fetal bovine serum, penicillin, and streptomycin. All cell lines were incubated in an atmosphere containing 5% CO2 at 37 oC. Materials. Primary antibodies against APP, acetyl-histone 3, histone 3, α-tubulin, acetyl-α-tubulin, Hsp90, HDAC6, acetyl-lysine were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against phospho-Tau (Ser396 and S404) were purchased from Abcam (Cambridge, MA, USA); antibodies to phospho-Tau (Ser396 and S404) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). An antibody against ubiquitin was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). A1-40 was purchased from AnaSpec (Fremont, CA, USA). Horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit IgG secondary antibodies were purchased from Jackson ImmunoResearch Inc. (West Grove, PA, USA). The pCAX FLAG APP and pRK5-EGFP-Tau P301L plasmids were provided by Dennis Selkoe and Tracy Young-Pearse (Addgene plasmid #30154) and Karen Ashe (Addgene plasmid #46908), respectively. TurboFect transfection reagent was from Fermentas (Burlington, Ontario, Canada). ACY-1215 was obtained from BioVision Inc. (Milpitas, CA, USA). Unless otherwise stated, all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Transfection assay. Cells were seeded one day before transfection. The plasmids pCAX FLAG APP and pRK5-EGFP-Tau P301L (1 g each) and 1 L of TurboFect transfection reagent were mixed for 20 min at room

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temperature, added to the cells, and the suspensions were incubated for 24 h at 37 oC in a humidified atmosphere containing 5% CO2. Flow cytometry. After drug treatment, the cells were collected, washed with cold phosphate-buffered saline (PBS), then fixed with 75% alcohol overnight at −20 oC. After centrifugation, the fixed cells were washed with cold PBS, resuspended in DNA extraction buffer (0.2 M Na2HPO4, 0.1 M citric acid, pH 7.8) for 30 min. The cells were centrifuged and incubated with propidium iodide (PI) (0.1% Triton X-100, 100 μg/mL RNase A, and 80 μg/mL PI in PBS) for 30 min. The cell cycle was analyzed using a FACScan Flow cytometer and Cell Quest software (Becton Dickinson, Mountain View, CA, USA). Immunoblot and immunoprecipitation analyses. Cells (1 × 106) were incubated for 10 min at 4 oC in lysis buffer (20 mM HEPES, pH 7.4, 2 mM EGTA, 50 mM β-glycerophosphate, 0.1% Triton X-100, 10% glycerol, 1 mM DTT, 1 μg/mL leupeptin, 5 μg/mL aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate), scraped from the plate, incubated on ice for 10 min, and centrifuged at 17,000 g at 4 oC for 30 min. Protein samples (20 μg) were electrophoresed through sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred onto a nitrocellulose membrane, which was then blocked by incubation at room temperature with 5% fat-free milk in PBS for 30 min. Immunoblotting was performed by incubating the membrane with primary antibodies in PBS overnight at 4 oC, followed by incubation with HRP-conjugated secondary antibodies at room temperature for 1 h. To measure bound antibodies, the membrane were


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trated with ECL reagent (T-Pro Biotechnology, New Taipei City, Taiwan), and exposed to a photographic film. Cell lysates (30 μg) were incubated with antibodies (1 μg each) overnight at 4 oC and protein A/G agarose beads. The precipitated beads were washed three times with 1 mL of ice-cold cell lysis buffer, and bound immune complexes were separated using 8% SDS-PAGE, followed by immunoblotting using the indicated primary antibody. Subcellular fractionation. This assay was carried out using a published method.20 Briefly, cells (1 × 107) were treated with drugs for 24 h and scraped off into breaking buffer (0.25M sucrose, 10 mM HEPES, pH 7.2, 1 mM MgAc2, and protease inhibitors). The lysate was centrifuged at 190,000 g for 1 h, and the supernatant was collected as the cytosolic fraction. The pellet was resuspended and incubated with 5 μM nocodazole on ice for 30 min and then centrifuged at 190,000 g for 1 h. The supernatant contained microtubule-tau, and the pellets contained membrane-bound and aggregated tau. The pellets were further extracted by 100 mM sodium carbonate buffer, pH 11.5, centrifuged at 190,000 g for 1 h, and washed with 1% SDS to produce a fraction containing tau aggregates. Samples containing equal amounts of protein were analyzed using SDS-PAGE. Analysis of cognitive dysfunction. Seven-week-old male Wistar rats were orally administered compound 6 (25 mg/kg) or vorinostat (SAHA, 50 mg/kg) with vehicle (1% carboxymethyl cellulose, 0.5% Tween 80, 0.1 mL/100 g) once daily for one week. The rats were then subjected to the Morris water maze and elevated plus maze tests,

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and the brain were removed for immunohistochemical stains. Female B6;129-Psen1tm1Mpm Tg(APPSwe,tauP301L)1Lfa/Mmjax (3xTg-AD) mice and control B6129SF2/J mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA), fed a standard diet for 6 months, then orally administered compound 6 (50 mg/kg) or memantine (30 mg/kg, dissolved in water) once daily for three months, and then subjected to the Morris water maze, and the brain were removed for immunohistochemical stains. Morris water maze. The water maze was constructed as a white circular pool (diameter, 150 cm; height, 65 cm; featureless inner-surface) filled with water to a height of 50 cm. A white platform (diameter, 10 cm; height, 45 cm) was submerged 5 cm below the water. Styrofoam beads were used to let the platform invisible, and the water temperature was adjusted to 23 oC–27 oC. The rats were trained by allowing to stay for 10 s upon reaching the platform. If the rat failed to locate the platform in 180 s, it was placed on the platform for 10 s to memorize the location of the platform. Training was performed twice daily and 4 days during 1 week. Test trials were repeated twice with a 20 min interval for each animal. Scopolamine (1 mg/kg) was administrated intraperitoneally to the animal 90 min before the test trial to induce AD-like features, except the control rats. Elevated plus maze. The plus maze included two open and two closed arms which connected by a central platform. Rats were placed at the end of the open arms facing away from the central platform. The time for each rat to


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move from the open arm to the closed arms was recorded. If the rat did not enter the closed arm within 180 s, it was gently pushed into the closed arm and assigned a transfer latency = 180 s. These experiments were carried out in accordance with relevant ethics guidelines and regulations, which were approved by the Animal Use and Management Committee of the College of Medicine, National Taiwan University (IACUC number: 20130361). Blood–brain barrier (BBB) crossing assay. This assay was performed by Eurofins Scientific. Briefly, 200–300 g SD rats were provided by BioLasco Taiwan (under the license of Charles River Laboratories). Rats were sedated by inhalation 3% isoflurane for blood collection using cardiac puncture 60 and 180 min after oral administration of the test compound. Aliquots of blood were gently mixed with lithium heparin, kept on ice, and centrifuged at 2,500 g for 15 min at 4 oC. The plasma was then harvested and stored at –70 oC. After blood sampling, rats were decapitated, and the brain was quickly removed, and rinsed with cold saline (0.9% NaCl, w/v). The surface vasculature was removed, blotted with dry gauze, weighed, and stored on ice within 1 h. Each brain was homogenized in 3 mL cold PBS, pH 7.4, for 10 s on ice, and centrifuged at 5400 g for 15 min at 4 oC. Supernatants were precipitated using acetonitrile precipitation and subjected to HPLCMS/MS. Data Analysis and Statistics.

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Each result represents the mean ± STD of at least three independent experiments. The data were analyzed using the Student’s t-test. One-way ANOVA was performed to analyze the animal data. Parameters with a p-value < 0.05 were considered statistically significant. Maximum tolerated dose assay The maximum tolerated dose (MTD) was determined in male balb/c mice at 100, 500, and 1000 mg/kg/day by intraperitoneal injection for 6 days and then recovered for 1 week. A group of six mice was injected with vehicle as control. Animals were monitored daily for body weight and survival. (C) Computational Study. Compound 6 was docked into the available crystal structures of HDAC1 (PDB ID: 5ICN), 2 (PDB ID: 5IX0), 3 (PDB ID: 4A69), 6 (PDB ID: 5EDU) and 8 (PDB ID: 1W22), and then compared the structures. The structures of HDAC1, 2, 3 and 8 were superimposed with that of HDAC6 using a structural alignment tool.31 The docking analysis was performed by the molecular docking software LeadIT.32 First, compound 6 was protonated in aqueous solution using the software. The HDAC isozyme binding site consisted of residues within a 12Å radius from the co-crystallized ligand. The hybrid enthalpy and entropy docking strategy was then applied. Finally, the parameters were used with default settings for the docking process.


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Supporting Information Available: The 1H and

13

C-NMR spectra for compounds 4-20. Full list of

molecular formula strings (CSV). This material is available free of charge via the Internet at http://pubs.acs.org.

Author Information. Corresponding Author *Liou J. P.: Phone: 886-2-2736-1661 ext 6130. E-mail: [email protected]. *Yang C. R.: Phone: 886-2-33668758. E-mail: [email protected]. ORCID: 0000-0001-5990-1346 ＆

Lee, H. Y., Fan, S. J. and Huang, F. I. contributed equally to this work

Acknowledgment. This research were supported by the Ministry of Science and Technology, Taiwan (grant no. MOST 106-2113-M-038-002, MOST 105-2320-B-038-010 and MOST 106-2320-B-002-006MY3).

ABBREVIATIONS USED

HDAC, histone deacetylases; AD, Alzheimer’s disease; NFTs, neurofibrillary tangles; SAHA, suberanilohydroxamic acid; HSP, heat shock protein; UPS, ubiquitin-proteasome system; BBB, bloodbrain-barrier; PDC, pyridinium dichromate; NH2OTHP, O-(tetrahydro-pyran-4-yl)-hydroxylamine;


47


EDC,

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide;

HOBt,

trifluoroacetic acid.


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hydroxybenzotriazole;

TFA,

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References 1. (a) Falkenberg, K. J.; Johnstone, R. W. Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders. Nat. Rev. Drug Discov. 2014, 13, 673–691.; (b) Fan, S. J.; Huang, F. I.; Liou, J. P.; Yang, C. R. The novel histone deacetylase 6 inhibitor, MPT0G211, ameliorates tau phosphorylation and cognitive deficits in an Alzheimer's disease model. Cell Death Dis. 2018, 9, 655.; (c) Rabal, O.;, Sánchez-Arias, J. A.; Cuadrado-Tejedor, M.; de Miguel, I.; PérezGonzález, M.; García-Barroso, C.; Ugarte, A.; Estella-Hermoso de Mendoza, A.; Sáez, E.; Espelosin, M.; Ursua, S.; Haizhong, T.; Wei, W.; Musheng, X.; Garcia-Osta, A.; Oyarzabal, J. Design, synthesis, biological evaluation and in vivo testing of dual phosphodiesterase 5 (PDE5) and histone deacetylase 6 (HDAC6)-selective inhibitors for the treatment of Alzheimer's disease. Eur. J. Med. Chem. 2018, 150, 506-524. 2. (a)Yang, Z.; Wang, T.; Wang, F.; Niu, T.; Liu, Z.; Chen, X.; Long, C.; Tang, M.; Cao, D.; Wang, X.; Xiang, W.; Yi, Y.; Ma, L.; You, J.; Chen, L. Discovery of selective histone deacetylase 6 inhibitors using the quinazoline as the cap for the treatment of cancer. J. Med. Chem. 2016, 59, 1455-1470. (b) Senger, J.; Melesina, J.; Marek, M.; Romier, C.; Oehme, I.; Witt, O.; Sippl, W.; Jung, M. Synthesis and biological investigation of oxazole hydroxamates as highly selective histone deacetylase 6 (HDAC6) inhibitors. J. Med. Chem. 2016, 59, 1545-1555. (c) Lin, X.; Chen, W.; Qiu, Z.; Guo, L.; Zhu, W.; Li, W.; Wang, Z.; Zhang, W.; Zhang, Z.; Rong, Y.; Zhang, M.; Yu, L.; Zhong, S.; Zhao, R.; Wu, X.; Wong, J. C.; Tang, G. Design and synthesis of orally bioavailable aminopyrrolidinone


49


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histone deacetylase 6 inhibitors. J. Med. Chem. 2015, 58, 2809-2820. (d) Lee, H. Y.; Tsai, A. C.; Chen, M. C.; Shen, P. J.; Cheng, Y. C.; Kuo, C. C.; Pan, S. L.; Liu, Y. M.; Liu, J. F.; Yeh, T. K.; Wang, J. C.; Chang, C. Y.; Chang, J. Y.; Liou, J. P. Azaindolylsulfonamides, with a more selective inhibitory effect on histone deacetylase 6 activity, exhibit antitumor activity in colorectal cancer HCT116 cells. J. Med. Chem. 2014, 57, 4009-4022. (e) Yu, C. W.; Chang, P. T.; Hsin, L. W.; Chern, J. W. Quinazolin-4-one derivatives as selective histone deacetylase-6 inhibitors for the treatment of Alzheimer's disease. J. Med. Chem. 2013, 56, 6775-6791. (f) Wagner, F. F.; Olson, D. E.; Gale, J. P.; Kaya, T.; Weïwer, M.; Aidoud, N.; Thomas, M.; Davoine, E. L.; Lemercier, B. C.; Zhang, Y. L.; Holson, E. B. Potent and selective inhibition of histone deacetylase 6 (HDAC6) does not require a surface-binding motif. J. Med. Chem. 2013, 56, 1772-1776. (g) Blackburn, C.; Barrett, C.; Chin, J.; Garcia, K.; Gigstad, K.; Gould, A.; Gutierrez, J.; Harrison, S.; Hoar, K.; Lynch, C.; Rowland, R. S.; Tsu, C.; Ringeling, J.; Xu, H. Potent histone deacetylase inhibitors derived from 4-(aminomethyl)N-hydroxybenzamide with high selectivity for the HDAC6 isoform. J. Med. Chem. 2013, 56, 72017211. (h) Kalin, J. H.; Butler, K. V.; Akimova, T.; Hancock, W. W.; Kozikowski, A. P. Secondgeneration histone deacetylase 6 inhibitors enhance the immunosuppressive effects of Foxp3+ Tregulatory cells. J. Med. Chem. 2012, 55, 639-651. (i) Bergman, J. A.; Woan, K.; Perez-Villarroel, P.; Villagra, A.; Sotomayor, E. M.; Kozikowski, A. P. Selective histone deacetylase 6 inhibitors bearing substituted urea linkers inhibit melanoma cell growth. J. Med. Chem. 2012, 55, 9891-9899. (k) Kozikowski, A. P.; Tapadar, S.; Luchini, D. N.; Kim, K. H.; Billadeau, D. D. Use of the nitrile


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oxide cycloaddition (NOC) reaction for molecular probe generation: a new class of enzyme selective histone deacetylase inhibitors (HDACIs) showing picomolar activity at HDAC6. J. Med. Chem. 2008, 51, 4370-4373. (l) Itoh, Y.; Suzuki, T.; Kouketsu, A.; Suzuki, N.; Maeda, S.; Yoshida, M.; Nakagawa, H.; Miyata, N. Design, synthesis, structure--selectivity relationship, and effect on human cancer cells of a novel series of histone deacetylase 6-selective inhibitors. J. Med. Chem. 2007, 50, 5425-5438. 3. (a) Santo, L.; Hideshima, T, Kung, A. L.; Tseng, J. C.; Tamang, D.; Yang, M.; Jarpe, M.; van Duzer, J. H.; Mazitschek, R.; Ogier, W. C.; Cirstea, D.; Rodig, S.; Eda, H.; Scullen, T.; Canavese, M.; Bradner, J.; Anderson, K. C.; Jones, S. S.; Raje, N. Preclinical activity, pharmacodynamic, and pharmacokinetic properties of a selective HDAC6 inhibitor, ACY-1215, in combination with bortezomib in multiple myeloma. Blood 2012, 119, 2579–2589. (b) www.clinicaltrials.gov (NCT01323751, Study of ACY-1215 Alone and in Combination With Bortezomib and Dexamethasone in Multiple Myeloma, accessed Mar 28, 2011) 4. Zhang, L.; Sheng, S.; Qin, C. The role of HDAC6 in Alzheimer’s disease. J. Alzheimers Dis. 2013, 33, 283–295. 5. Stokin, G. B.; Goldstein, L. S. Axonal transport and Alzheimer's disease. Annu. Rev. Biochem. 2006; 75, 607–627.


51


Page 52 of 57

6. Butler, K. V.; Kalin, J.; Brochier, C.; Vistoli, G.; Langley, B.; Kozikowski, A. P. Rational design and simple chemistry yield a superior, neuroprotective HDAC6 inhibitor, tubastatin A. J. Am. Chem. Soc. 2010, 132, 10842–10846. 7. Majid, T.; Griffin, D.; Criss, II, Z.; Jarpe, M.; Pautler, R. G. Pharmocologic treatment with histone deacetylase 6 inhibitor (ACY-738) recovers Alzheimer’s disease phenotype in amyloid precursor protein/presenilin 1 (APP/PS1) mice. Alzheimer’s & Dementia: Translational Research & Clinical Interventions 2015, 1, 170–181. 8. Bertrand, P. Inside HDAC with HDAC inhibitors. Eur. J. Med. Chem. 2010, 45, 2095–2116. 9. Mahboobi, S.; Sellmer, A.; Höcher, H.; Garhammer, C.; Pongratz, H.; Maier, T.; Ciossek, T.; Beckers, T. 2-Aroylindoles and 2-aroylbenzofurans with N-hydroxyacrylamide substructures as a novel series of rationally designed histone deacetylase inhibitors. J. Med. Chem. 2007, 50, 4405– 4418. 10. Ragno, R.; Mai, A.; Massa, S.; Cerbara, I.; Valente, S.; Bottoni, P.; Scatena, R.; Jesacher, F.; Loidl, P.; Brosch, G. 3-(4-Aroyl-1-methyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamides as a new class of synthetic histone deacetylase inhibitors. 3. Discovery of novel lead compounds through structurebased drug design and docking studies. J. Med. Chem. 2004, 47, 1351–1359. 11. Liou, J. P.; Wu, C. Y.; Hsieh, H. P.; Chang, C. Y.; Chen, C. M.; Kuo, C. C.; Chang, J. Y. 4- and 5aroylindoles as novel classes of potent antitubulin agents. J. Med. Chem. 2007, 50, 4548–4552.


52

Page 53 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


12. Amand, R.; Gill, K. D.; Mahdi, A. A. Therapeutics of Alzheimer’s disease: past, present and future. Neuropharmacology 2014, 76, 27–50. 13. Barghorn, S.; Zheng-Fischhofer, Q.; Ackmann, M.; Biernat, J.; von Bergen, M.; Mandelkow, E. M.; Mandelkow, E. Structure, microtubule interactions, and paired helical filament aggregation by tau mutants of frontotemporal dementias. Biochemistry 2000, 39, 11714–11721. 14. Roberson, E. D.; Scearce-Levie, K.; Palop, J. J.; Yan, F.; Cheng, I. H.; Wu, T,; Gerstein, H.; Yu, G. Q.; Mucke, L. Reducing endogenous tau ameliorates amyloid β-induced deficits in an Alzheimer’s disease mouse model. Science 2007, 316, 750–754. 15. Govindarajan, N.; Rao, P.; Burkhardt, S.; Sananbenesi, F.; Schluter, O. M.; Bradke, F.; Lu, J.; Fischer, A. Reducing HDAC6 ameliorates cognitive deficits in a mouse model for Alzheimer’s disease. EMBO. Mol. Med. 2013, 5, 52–63. 16. Young-Pearse, T. L.; Bai, J.; Chang, R.; Zheng, J. B.; LoTurco, J. J.; Selkoe, D. J. A critical function for beta-amyloid precursor protein in neuronal migration revealed by in utero RNA interference. J. Neurosci. 2007, 27, 14459–14469. 17. Hoover, B. R.; Reed, M. N.; Su, J.; Penrod, R. D.; Kotilinek, L. A.; Grant, M. K.; Pitstick, R.; Carlson, G. A.; Lanier, L. M.; Yuan, L. L.; Ashe, K. H.; Liao, D. Tau mislocalization to dendritic spines mediates synaptic dysfunction independently of neurodegeneration. Neuron 2010, 68, 1067– 1081.


53


Page 54 of 57

18. Simic, G.; Babic Leko, M.; Wray, S.; Harrington, C.; Delalle, I.; Jovanov-Milosevic, N.; Bazadona, D.; Buee, L.; de Siva, R.; Di Giovanni, D.; Wischik, C.; Hof, P. R. Tau protein hyperphosphorylation and aggregation in Alzheimer’s disease and other tauopathies, and possible neuroprotective strategies. Biomolecules 2016, 6, 6. 19. Nisbet, R. M.; Polanco, J. C.; Ittner, L. M.; Götz, J. Tau aggregation and its interplay with amyloid-β. Acta Neuropathol. 2015, 129, 207–220. 20. Dou, F.; Netzer, W. J.; Tanemura, K.; Li, F.; Hartl, F. U.; Takashima, A.; Gouras, G. K.; Greengard, P.; Xu, H. Chaperones increase association of tau protein with microtubules. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 721–726. 21. Sulistio, Y. A.; Heese K. The ubiquitin-proteasome system and molecular chaperone deregulation in Alzheimer’s disease. Mol. Neurobiol. 2016, 53, 905–931. 22. Dickey, C. A.; Dunmore J.; Lu, B.; Wang, J. W.; Lee, W. C.; Kamal, A.; Burrows, F.; Eckman, C.; Hutton, M.; Petrucelli, L. HSP induction mediates selective clearance of tau phosphorylated at proline-directed Ser/Thr sites but not KXGS (MARK) sites. FASEB J. 2006, 20, 753–755. 23. Cook, C.; Gendron, T. F.; Scheffel, K.; Carlomagno, Y.; Dunmore, J.; DeTure, M.; Petrucelli, L. Loss of HDAC6, a novel CHIP substrate, alleviates abnormal tau accumulation. Hum. Mol. Genet. 2012, 21, 2936–2945. 24. Kamal, A.; Boehm, M. F.; Burrows, F. J. Therapeutic and diagnostic implications of Hsp90 activation. Trends Mol. Med. 2004, 10, 283–290.


54

Page 55 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


25. Scroggins, B. T.; Robzyk, K.; Wang, D.; Marcu, M. G.; Tsutsumi, S.; Beebe, K.; Cotter, R. J.; Felts, S.; Toft, D.; Karnitz, L.; Rosen, N.; Neckers, L. An acetylation site in the middle domain of Hsp90 regulates chaperone function. Mol. Cell 2007, 25, 151–159. 26. Kovacs, J. J.; Murphy, P. J.; Gaillard, S.; Zhao, X.; Wu, J. T.; Nichitta, C. V.; Yoshida, M.; Toft, D. O.; Pratt, W. B.; Yao, T. P. HDAC6 regulates Hsp90 acetylation and chaperone-dependent activation of glucocorticoid receptor. Mol. Cell 2005, 18, 601–607. 27. Cook, C.; Gendron, T. F.; Scheffel, K.; Carlomagno, Y.; Dunmore, J.; DeTure, M.; Petrucelli, L. Loss of HDAC6, a novel CHIP substrate, alleviates abnormal tau accumulation. Hum. Mol. Genet. 2012, 21, 2936–2945. 28. Dou, F.; Netzer, W. J.; Tanemura, K.; Li, F.; Hartl, U.; Takashima, A.; Gouras, G. K., Greengard, P.; Xu, H. Chaperones increase association of tau protein with microbutules. Proc. Natl. Acad. Sci. U.S. A. 2003, 100, 721–726. 29. Martinez-Coria, H.; Green, K. N.; Billings, L. M.; Kitazawa, M.; Albrecht, M.; Rammes, G.; Parsons, C. G.; Gupta, S.; Banerjee, P.; LaFerla, F. M. Memantine improves cognition and reduces Alzheimer’s-like neuropathology in transgenic mice. Am. J. Pathol. 2010, 176, 870–880.

30. Odagiri, S.; Tanji, K.; Mori, F.; Miki, Y.; Kakita, A.; Takahashi, H.; Wakabayashi, K. Brain expression level and activity of HDAC6 protein in neurodegenerative dementia. Biochem. Biophys. Res. Commun. 2013, 430, 394–399.


55


Page 56 of 57

31. Shindyalov, I. N.; Bourne, P. E. Protein structure alignment by incremental combinatorial extension (CE) of the optimal path. Protein Engineering 1998, 11, 739–747. 32. LeadIT, B. http://www. biosolveit. de/LeadIT. accessed 12th Mar 2011.


56

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5-Aroylindoles Act as Selective Histone Deacetylase 6 Inhibitors

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